Although adenosine signaling is involved in numerous cellular functions by engaging its membrane receptors, little is known about its role in erythrocytes. The goal of this Project is to address the major theme of this PPG by focusing on the role of the erythrocyte in the hypoxic adenosine response in normal individuals and those with sickle cell disease (SCD); a disease constantly facing hypoxia with dangerous complications including pulmonary hypertension (PH) and kidney damage. The proposed research builds on our recent discovery that elevated adenosine signaling through the A2B adenosine receptor (AD0RA2B) promotes sickling, a key feature of the disease, by the induction of 2,3-diphosphoglycerate (2,3-DPG), an erythroid specific molecule known to decrease hemoglobin (Hb) O2 binding affinity. Intriguingly, extending the findings from SCD, we discovered that inhibition of equlibrative nucleoside transporters (ENTs) coupled with AD0RA2B also induces 2,3-DPG levels in normal erythrocytes. Thus, our studies raise a novel and compelling hypothesis that increased adenosine is beneficial for normal individuals by facilitating O2 release to prevent acute ischemic injury. However, this process is detrimental for SCD patients by promoting O2 release, deoxyHbS polymerization, sickling and tissue injury. To test this hypothesis, three specific aims are proposed. AIM I. Extend our discovery of detrimental effects of elevated adenosine signaling in SCD to preclinical animal studies and human translational studies. We will use SCD Berkley mice as an investigative tool to test the efficacy of lowering adenosine by enzyme therapy, or inhibiting AD0RA2B signaling by specific antagonists, on morbidity and mortality. Moreover, we will collaborate with NHLBI to analyze 236 adult samples obtained from a large cohort of SCD patients to determine whether elevated adenosine and 2,3-DPG levels are novel pathogenic biomarkers correlated to disease severity and PH. AIM II. Define the importance of AD0RA2B signaling in normal and SCD erythrocytes in hypoxia-induced tissue injury. We will generate a novel genetic mouse line with the deletion of erythrocyte-specific AD0RA2B to define the function of erythrocytes in ischemic injury under normal and SCD conditions. AIM III. Evaluate the role of ENT on normal and SCD erythrocytes in the hypoxic adenosine response. We will use both pharmacological and novel genetic approaches to assess the contribution of erythrocyte ENTs to blood adenosine levels, O2 binding and hypoxic injury under normal and SCD settings. Overall, our findings will provide new insight on the role of the erythrocyte in the hypoxic adenosine response. RELEVANCE (See instructions): In sickle cell disease (SCD), hemoglobin mutations result in near constant hypoxia. We recently found a molecule that is detrimental for SCD by promoting O2 release, sickling, and tissue injury. However, this molecule is likely beneficial for normal individuals by stimulating O2 release to prevent hypoxic injury. Our findings could have a significant impact on the management of acute ischemic injury in normal individuals and chronic responses in SCD patients.